A histidine/tryptophan pi-stacking interaction stabilizes the heme-independent folding core of microsomal apocytochrome b5 relative to that of mitochondrial apocytochrome b5

The outer mitochondrial membrane isoform of mammalian cytochrome b5 (OM b5) is considerably more stable than its microsomal counterpart (Mc b5), whereas the corresponding apoproteins (OM and Mc apo-b5) exhibit similar stability. OM and Mc apo-b5 are also similar in that their empty heme-binding pockets (core 1) are highly disordered but that the remainder of each apoprotein (core 2) displays substantial hololike structure. Core 1 residue 71 is leucine in all known mammalian OM b5's and serine in the corresponding Mc proteins. Replacing Leu-71 in rat OM (rOM) b5 with Ser has been shown to (1) decrease apoprotein thermodynamic stability by >2 kcal/mol and (2) extend conformational disorder beyond core 1 and into core 2, as evidenced in part by loss of a near-UV circular dichroism signal associated with the side chain of invariant residue Trp-22. Herein we report identification of a conserved Mc b5 core 2 packing motif that plays a key role in stabilizing apoprotein conformation in the vicinity of Trp-22, thereby compensating for the presence of Ser at position 71: a pi-stacking interaction between the side chains of Trp-22 and His-15 that is extended by hydrogen bonding between the side chains of His-15, Ser-20, and Glu-11. The corresponding conserved packing motif in OM b5's differs in having arginine at position 15 and glutamate at position 20. We also present evidence indicating that the conserved Mc b5 packing motif noted above contributes to the unusually extensive secondary structure exhibited by bovine Mc apo-b5 in the urea-denatured state.     

Backbone dynamics of apocytochrome b5 in its native, partially folded state

The backbone dynamics in the native state of apocytochrome b5 were studied using 15N nuclear magnetic spin relaxation measurements. The field (11.7 and 14.1 T) and temperature (10-25 degrees C) dependence of the relaxation parameters (R1, R2, and R1rho) and the 1H-15N NOE established that the protein undergoes multiple time scale internal motions related to the secondary structure. The relaxation data were analyzed with the reduced spectral density mapping approach and within the extended model-free framework. The apoprotein was confirmed to contain a disordered heme-binding loop of approximately 30 residues with dynamics on the sub-nanosecond time scale (0.6 < S2 < 0.7, 100 ps < taue < 500 ps). This loop is attached to a structured hydrophobic core, rigid on the picosecond time scale (S2 > 0.75, taue < 50 ps). The inability to fit the data for several residues with the model-free protocol revealed the presence of correlated motion. An exchange contribution was detected in the transverse relaxation rate (R2) of all residues. The differential temperature response of R2 along the backbone supported slower exchange rates for residues in the loop (tauex > 300 micros) than for the folded polypeptide chain (tauex < 150 micros). The distribution of the reduced spectral densities at the 1H and 15N frequencies followed the dynamic trend and predicted the slowing of the internal motions at 10 degrees C. Comparison of the dynamics with those of the holoprotein [Dangi, B., Sarma, S., Yan, C., Banville, D. L., and Guiles, R. D. (1998) Biochemistry 37, 8289-8302] demonstrated that binding of the heme alters the time scale of motions both in the heme-binding loop and in the structured hydrophobic core.     

Thermodynamics of apocytochrome b5 unfolding.

Apocytochrome b5 from rabbit liver was studied by scanning calorimetry, limited proteolysis, circular dichroism, second derivative spectroscopy, and size exclusion chromatography. The protein is able to undergo a reversible two-state thermal transition. However, transition temperature, denaturational enthalpy, and heat capacity change are reduced compared with the holoprotein. Apocytochrome b5 stability in terms of Gibbs energy change at protein unfolding (delta G) amounts to delta G = 7 +/- 1 kJ/mol at 25 degrees C (pH 7.4) compared with delta G = 25 kJ/mol for the holoprotein. Apocytochrome b5 is a compact, native-like protein. According to the spectral data, the cooperative structure is mainly based in the core region formed by residues 1-35 and 79-90. This finding is in full agreement with NMR data (Moore, C.D. & Lecomte, J.T.J., 1993, Biochemistry 32, 199-207).     

Structural propensities in the heme binding region of apocytochrome b5. II. Heme conjugates

In the absence of heme cofactor, the water-soluble domain of rat microsomal cytochrome b5 (cyt b5) contains a long flexible region within its 42-residue heme-binding loop. Heme capture induces this region to fold into a well-defined structure containing helices H3-H5, each separated by a turn, with His39 and His63 serving as axial ligands to the heme iron. We have shown that the H4 region of the apoprotein has the greatest tendency for disorder within the isolated binding loop. Here, the effect of the His63-iron bond and proximity of heme plane on the population of helical conformation in H4 and H5 was investigated by synthesis and characterization of a peptide-sandwiched mesoheme construct in which two H4-H5 peptides were covalently attached to a single cofactor. Spectroscopic data indicated that a holoprotein-like bis-histidine coordination state was achieved over a pH range from 7 to 9. Trifluoroethanol titrations of the construct and the analogous free peptide under these pH conditions revealed that heme proximity and iron ligation were insufficient to promote helix formation in H4 and H5. These observations were used to assess the role of disordered regions in heme capture and the loop-scaffold interface in holoprotein folding and stability.     

Structural propensities in the heme binding region of apocytochrome b5. I. Free peptides

The water-soluble domain of rat microsomal cytochrome b5 is a small globular hemoprotein. Under native conditions, the apoprotein consists of a well-folded hydrophobic core and a 42-residue loop, which is substantially disordered in solution. Association with the heme cofactor causes the loop to organize into a second well-folded hydrophobic core encompassing four short helices, H2-H5. Of these, H3 and H4 are recognized as intrinsically disordered by algorithms that analyze primary structures for folding propensities. Three peptides, spanning H2-H5, H2-H3, and H4-H5, were designed, synthesized, and characterized to identify local structural preferences in the isolated loop. In addition, two replacements (D60R and N57P, which are known to stabilize holocytochrome b5) were introduced individually in the H4-H5 peptide. Helical content measured by nuclear magnetic resonance and far-UV circular dichroism spectroscopy in solutions of 2,2,2-trifluoroethanol revealed that H4 possessed a lower propensity to form the holoprotein structure than H3. Both replacements in H4 resulted in measurable changes in observed overall helical propensities. It was concluded that the prediction of intrinsic disorder was reliable. Furthermore, the stability of the holoprotein did not correlate simply with helical propensities in the disordered regions.     

Modification of trypsin-solubilized cytochrome b5, apocytochrome b5, and liposome-bound cytochrome b5 by diethylpyrocarbonate

The interactions of diethylpyrocarbonate (DEP) with the various forms of cytochrome b5 were studied to gain a better understanding of the factors that influence the extent of modification of the axial histidines of cytochrome b5. Very low concentrations of DEP were able to decrease the heme binding capacity of apocytochrome b5. Moreover, it was shown that two additional histidines, presumed to be the axial ligands (His 39 and 63), were modified in the apo but not the holo form of a given preparation of cytochrome b5. Trypsin-solubilized bovine cytochrome b5 was resistant to the effects of DEP. A 200-fold molar excess of DEP displaced only 15% of the heme in the trypsin-solubilized protein in contrast to an 84% displacement of the heme in the detergent-solubilized protein. However, detergent-solubilized cytochrome b5 which had been incorporated into phospholipid vesicles exhibited the same reactivity with DEP as did the trypsin-solubilized protein. This is attributed to the fact that the two resistant preparations of cytochrome b5 are monomeric in their respective environments while detergent-solubilized cytochrome b5 is known to exist as an octamer in aqueous solutions. Our studies suggest that dissociation of the octamer to the monomer results in a conformational change that decreases the reactivity of the axial ligands of the hydrophilic heme-containing domain of cytochrome b5. Examination of the cytochrome b5 molecule by computer graphics indicates that a tunnel leads from the surface of the molecule to axial histidine 63 and that axial histidine 39 is buried.     

Similarities in structure between holocytochrome b5 and apocytochrome b5: NMR studies of the histidine residues

The properties of the six histidine residues of apocytochrome b5 have been investigated by using one- and two-dimensional proton NMR spectroscopy in order to probe the structure remaining after heme removal. Spectral assignments were arrived at by analyzing proton NOE connectivities, comparing them to those observed in the holoprotein, and inspecting the X-ray structure of the latter species. Each histidine residue was studied for its pKa value, interaction with the relaxation agent copper nitrilotriacetic acid, and reactivity toward bromoacetic acid. The four histidines which are not coordinated to the iron atom in the holoprotein (His-15, -26, -27, and -80) display in the major conformer of the apoprotein the same characteristic properties as in the holoprotein. Three of them are involved in specific interactions with the rest of the structure: His-15 and His-80 participate in hydrogen bonds, and His-27 is influenced by the nearby C-terminal segment. His-26 is the most exposed to the solvent. His-63 and His-39, which are located in the heme binding site, have distinct pKa values; they are affected differently by the copper agent and exhibit comparable reactivity toward bromoacetic acid, albeit milder than that of His-26. The results show that the heme binding residues are clearly distinguishable by their physicochemical properties and that several elements of native holoprotein structure are in place in the apoprotein. It is proposed that the structural influence of the heme is localized and that the amino- and carboxy-terminal segments form a structural unit providing stability to the apoprotein and supporting a fluctuating, partially folded binding site.     

Repacking of hydrophobic residues in a stable mutant of apocytochrome b562 selected by phage-display and proteolysis

To test a hydrophobic core-directed protein design approach, we previously have used phage-display and proteolysis to select stably folded proteins from a library of mutants of apocytochrome b562. The consensus sequence of the selected mutants has hydrophilic residues at two of the three positions that are designed to form a hydrophobic core. To understand this unexpected result, we determined the high-resolution structure of one of the selected mutants using multi-dimensional nuclear magnetic resonance (NMR). The structure shows that the two hydrophilic residues in the consensus sequence were on the surface of the structure. Instead, two of their neighboring hydrophobic residues reorganized their side-chain conformations and formed the hydrophobic core. This result suggests that the hydrophobic core-directed protein design by phage-display and proteolysis is a valid method in general but alternative hydrophobic packing needs to be considered in the initial design. The unexpected repacking of the hydrophobic residues also highlights the plastic nature of protein structures.     

Structural and ion-binding properties of an S100b protein mixed disulfide: comparison with the reappraised native S100b protein properties

S100b protein, chemically modified by thioethanol groups (linked via disulfide bonds to two out of four Cys per dimer) was largely similar to reduced native S100b protein in its overall structure and differed only by small modifications extending, however, to the whole protein structure. Studies combining direct Ca2+ binding and associated conformational changes revealed that this chemical modification markedly increased the Ca2(+)-binding affinities (especially in the presence of physiological concentrations of K+ and Mg2+) and introduced a strong positive cooperativity. Different binding models are discussed and it emerges that in both proteins the Ca2(+)-binding sites are not equivalent and probably interact. Like the reduced protein, chemically modified S100b protein binds four Zn2+ ions in two classes of sites (of high and low affinities). Whereas the overall Zn2+ affinity was only slightly decreased, the binding sequence was probably reversed by the introduction of thioethanol groups. Moreover, in the presence of zinc, the Ca2+ affinities were higher and even identical, in both proteins.     

Structural analysis of the lipopolysaccharide derived core oligosaccharides of Actinobacillus pleuropneumoniae serotypes 1, 2, 5a and the genome strain 5b

The structures of the core oligosaccharides of the lipopolysaccharides (LPS) from Actinobacillus pleuropneumoniae serotypes 1, 2, 5a and 5b were elucidated. The LPS's were subjected to a variety of degradative procedures. The structures of the purified products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structures for the core oligosaccharides were determined on the basis of the combined data from these experiments. [carbohydrate formula see text] For serotype 1: R is (1S)-GalaNAc-(1-->4,6)-alpha-Gal II-(1-->3)-beta-Gal I-(1-->, and R' is H For serotype 2: R is beta-Glc III-(1-->, and R' is D-alpha-D-Hep V-(1--> For serotypes 5a and 5b: R is H and R' is D-alpha-D-Hep V-(1--> All oligosaccharides elaborated a conserved inner core structure, as illustrated. All sugars were in the pyranose ring form apart from the open-chain N-acetylgalactosamine, the identification of which in the serotype 1 LPS was of interest.     

Defining folding and unfolding reactions of apocytochrome b5 using equilibrium and kinetic fluorescence measurements.

The refolding and unfolding kinetics of a soluble domain of apocytochrome b5 extending from residue 1 to 104 have been characterized using stopped flow and equilibrium-based fluorescence methods. The isolated apoprotein unfolds reversibly in the presence of GuHCl. From cooperative unfolding curves, the conformational stability (Delta G(uw)), in the absence of denaturant, is estimated to be 11.6 +/- 1.5 kJ mol-1 at 10 degrees C. The stability of apocytochrome b5 is lower than that of the corresponding form of the holoprotein (Delta G approximately 25 kJ mol-1) and exhibits a transition midpoint at 1.6 M GuHCl. Kinetic studies support the concept of a two-state model with both unfolding and refolding rates showing an exponential dependence on denaturant concentration with no evidence of the formation of transient intermediates in either limb of the chevron plot. Apocytochrome b5 is therefore an example of a protein in which both kinetics and equilibria associated with folding are described by a two-state model. The values of mku and mkf obtained from kinetic analysis are an indication of a transition state (mku/meq of 0.29) that resembles the native form by retaining similar solvent accessibility and many of the noncovalent interactions found in the apoprotein. The changes in heat capacity support a transition state that resembles the apoprotein with a value for Delta Cpf of -3.6 kJ mol-1 K-1 estimated for the refolding reaction. From these measurements, a model of refolding that involves the rapid nucleation of hydrophobic residues around Trp26 is suggested as a major event in the formation of the native apoprotein.     

Pet127 governs a 5' -> 3'-exonuclease important in maturation of apocytochrome b mRNA in Saccharomyces cerevisiae

The details of mRNA maturation in Saccharomyces mitochondria are not well understood. All seven mRNAs are transcribed as part of multigenic units. The mRNAs are processed at a common 3'-dodecamer sequence, but the 5'-ends have seven different sequences. To investigate whether apocytochrome b (COB) mRNA is processed at the 5'-end from a longer precursor by an endonuclease or an exonuclease, a 64-nucleotide sequence, which is required for the protection of COB mRNA by the Cbp1 protein and is found at the 5'-end of the processed COB mRNA, was duplicated in tandem. The wild-type 64-nucleotide element functioned in either the upstream or downstream position when paired with a mutant element. In the tandem wild-type strain, the 5'-end of the mRNA was at the 5'-end of the upstream unit, demonstrating that the mRNA is processed by an exonuclease. Accumulation of precursor COB RNA in single and double element strains with a deletion of PET127 demonstrated that the encoded protein governs the 5'-exonuclease responsible for processing the precursor to the mature form.     

The identification of apocytochrome b as a mitochondrial gene product and immunological evidence for altered apocytochrome b in yeast strains having mutations in the COB region of mitochondrial DNA.

The yeast mitochondrial translation product of Mr 30 000 is identical with apocytochrome b. After labelling in vivo with [35S]sulphate in the presence of cycloheximide, the radioactivity in this product present in solubilized submitochondrial particles, was completely recovered in pure cytochrome bc1 complex as a single polypeptide. We show that this translation product is identical with apocytochrome b using peptide mapping by limited proteolysis according to Cleveland et al. [J. Biol. Chem. 250 (1977) 8236-8242] and by immunoprecipitation with a specific antiserum against apocytochrome b. New mitochondrial translation products in 36 strains of Saccharomyces cerevisiae having mutations in the COB region of the mitochondrial DNA, are precipitated by this antiserum. This is consistent with the assumption that many of the cob mutations are localized in the structural gene for apolcytochrome b on mitochondrial DNA. Mutations in two intervening sequences can give rise to products related to apocytochrome b that are considerably longer than normal apocytochrome b. We discuss the hypothesis that in these mutants splicing of the messenger RNA does not occur correctly and that, as a consequence of this, ribosomes read through in an intervening sequence.     

The group I intron of apocytochrome b gene from Chlamydomonas smithii encodes a site-specific endonuclease

The mitochondrial DNA molecules of two interfertile algal species, Chlamydomonas smithii and C. reinhardtii, are co-linear except for a 1075 bp intron (the -insert) that is present in the cob gene of C. smithii. The -insert, a group I intron (Cs cob·1) containing an open reading frame (ORF) which encodes a basic, hydrophilic protein of 237 amino acids, is unidirectionally transmitted to all diploid progeny during interspecific crosses. In this report, we show that the Cs cob·1-encoded protein is a site-specific endonuclease (I-Csm I) which could mediate the intron transfer via the gene conversion mechanism. The Cs cob·1 ORF was cloned into the vector pMALcr1 and over-expressed as a hybrid protein fused to maltose-binding protein (MBP). This fusion protein exhibited an in vivo endonuclease activity which specifically cleaved the intron homing site within the intronless cob gene.     

A test of the relationship between sequence and structure in proteins: excision of the heme binding site in apocytochrome b5.

The water-soluble domain of rat hepatic holocytochrome b5 is an alphabeta protein containing elements of secondary structure in the sequence beta1-alpha1-beta4-beta3-alpha2-alpha3-beta5- alpha4-alpha5-beta2-alpha6. The heme group is enclosed by four helices, a2, a3, a4, and a5. To test the hypothesis that a small b hemoprotein can be constructed in two parts, one forming the heme site, the other an organizing scaffold, a protein fragment corresponding to beta1-alpha1-beta4-beta3-lambda-beta2-alpha6 was prepared, where lambda is a seven-residue linker bypassing the heme binding site. The fragment ("abridged b5") was found to contain alpha and beta secondary structure by circular dichroism spectroscopy and tertiary structure by Trp fluorescence emission spectroscopy. NMR data revealed a species with spectral properties similar to those of the full-length apoprotein. This folded form is in slow equilibrium on the chemical shift time scale with other less folded species. Thermal denaturation, as monitored by circular dichroism, absorption, and fluorescence spectroscopy, as well as size-exclusion chromatography-fast protein liquid chromatography (SEC-FPLC), confirmed the coexistence of at least two distinct conformational ensembles. It was concluded that the protein fragment is capable of adopting a specific fold likely related to that of cytochrome b5, but does not achieve high thermodynamic stability and cooperativity. Abridged b5 demonstrates that the spliced sequence contains the information necessary to fold the protein. It suggests that the dominating influence to restrict the conformational space searched by the chain is structural propensities at a local level rather than internal packing. The sequence also holds the properties necessary to generate a barrier to unfolding.